CN107079817A - Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method - Google Patents
Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method Download PDFInfo
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- CN107079817A CN107079817A CN201710472440.0A CN201710472440A CN107079817A CN 107079817 A CN107079817 A CN 107079817A CN 201710472440 A CN201710472440 A CN 201710472440A CN 107079817 A CN107079817 A CN 107079817A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- Developmental Biology & Embryology (AREA)
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Abstract
Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method is related to the Plant Tissue Breeding mating system of bioengineering, comprises the following steps：A, artificial hybridization pollination；B, aseptic seeding and protocorm sprout the stage；C, Proliferation, Differentiation cultivation stage；D, strengthening seedling and rooting stage；E, hardening and transplanting." excellent pocket blue " combines the merit of parents, compact with plant type, and flower-shape is unique elegant, and pattern is splendid beautiful, it is easy to cultivate, the advantages of average florescence is up to 50 days, it is to avoid spend the long easy lodging of several single and scape and the shortcoming fractureed.Pass through the inventive method; seed germination rate and flowering rate are high; cultivation period is 34 years; it is the high efficiency method for test tube propagation for obtaining a large amount of " excellent pocket is blue " seedlings, high with germination rate, seedling is fast; the advantage such as seedling quality better, growth potential be strong; ordered one test tube seedling can be obtained, and substantial amounts of seedling is provided in a short time, so as to preferably protect wild resource.
Description
Technical field
The present invention relates to the Plant Tissue Breeding mating system of bioengineering, more particularly to a kind of Lanzhou-Xinjiang kind of pocket is " excellent
The tissue culture and rapid propagation method of excellent pocket orchid ".
Background technology
Orchid is one of maximum family of nature, is the hugest and high class of Evolutionary Levels in flowering plant
One of group, is only second to composite family among angiosperm and grass family is located at the third-largest section.Global orchid there are about 700
Category, 20000-30000 kinds are distributed widely in the broad area from temperate zone, subtropical zone to the torrid zone.The orchid that China has found and named
The category of flower about more than 170 more than 1300 is planted, wherein 2/3 is epiphytic orchid, 1/3 is Terrestrial orchid.At present, whole orchid has been put into
《CITS》Annex I or II.Guizhou is the plateau province of a karst landform extensive development
Part, subtropical climate is belonged on geographical environment, with the various and good hydro-thermal of unique complicated topography and geomorphology, vegetation pattern
Condition, suitable various plants are multiplied and lived, while also having bred more rich orchid resource.Orchid family plant is distributed with Guizhou Province within the border
The category of thing about 76 more than 320 is planted, including substantial amounts of mutation and kind.
Paphiopedilum（Paphiopedilum Pfitz）Plant is one of species of orchid most ornamental value, and pocket is blue
Due to unique flower moulding, gorgeous flower color, it is lasting view and admire the florescence and with high ornamental value, the whole world is wide
General cultivation.China is one of main place of production of cypripedium, be world's Paphiopedilum important distribution and many novel species mould
The formula place of production, there is 18 kinds（Chen, 1999）.From in the early 1980s, the cypripedium of China begins to appear in European ＆ American Market；
The end of the eighties to the mid-90 is the peak of China's Wild Paphiopedilum platymiscium trade, and pocket orchid market expands rapidly, countless
Plant appear in some American-European plantations and flowers market.
Pocket Lanzhou and Xinjiang kind " excellent "（ Paphiopedilum GZSLKY Youyou）, on 08 17th, 2016
The Royal Horticultural Society（Royal Horticultural Society abbreviations RHS）Log in, and obtain orchid new varieties and step on
Remember certificate.The Royal Horticultural Society is that unique, authority orchid new varieties log in mechanism in the world, and the mechanism only logs in head
Secondary incubation orchid category between and interspecific hybrid." excellent pocket is blue " is blue with long valve pocket（Paphiopedilum dianthum）For
Female parent, leaflet pocket is blue（Paphiopedilum barbigerum）For male parent, the method seed selection of being cross-breeding is formed,
Due to " excellent pocket blue " embryonic development not exclusively, breed difficult in its natural state, it is extremely low to there is seed germination rate, planting percent
It is low, the shortcomings of speed of growth is slow；Traditional ramificatiom breeding method, the cycle is long, and breeding coefficient is low, can only breed 1~3 every year
Times, Sterile culture difficulty is big.Therefore how to facilitate method quickly to breed " excellent pocket is blue " with plant biological has turned into orchid family plant
Thing child care and the emphasis problem of application.
The crossbreeding of Paphiopedilum by Germination Condition due to being limited, and development is very slow, since orchid is sterile
After seeding technique application, cypripedium crossbreeding is developed rapidly.But " excellent pocket is blue " belongs to orchid family, Paphiopedilum and planted
The new varieties of thing, its tissue culture and rapid propagation method is still in groping, processing stage, and " excellent pocket is blue " seed is extremely small, using other
The ripe tissue-culturing rapid propagation method for culturing seedlings of Paphiopedilum, causes abortion rate high, or even the state of stamen abortion occur.And orchid
Cultivation period is again long, only hybridization, nursery stage easily will 1-3.
So study a kind of abortion rate it is low, using tissue culture and rapid propagation method, volume facility method for culturing seedlings can be achieved, just turn into
" excellent pocket is blue " quickly moves towards market, meets the important prerequisite condition of people's demand.
The content of the invention
To solve " excellent pocket is blue " in the prior art, because embryonic development is incomplete, the technology that difficulty is bred in its natural state is asked
A kind of topic, abortion rate of present invention offer is low, can greatly improve the high efficiency method for test tube propagation of breeding coefficient, i.e. pocket Lanzhou-Xinjiang
Kind " excellent pocket is blue " tissue culture and rapid propagation method.
Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method, comprises the following steps：
A, artificial hybridization pollination：It is female parent with long valve pocket orchid, leaflet pocket orchid is male parent, the long valve pocket for choosing robust growth is blue female
This and male parent carry out artificial supplementary pollination when blooming, 370 to 390 days after pollination, will be developed to and are mature on the whole and uncracked
Fruit is used as explant；
B, aseptic seeding and protocorm sprout the stage：From full fruit with alcohol-pickled, it is placed in mercuric chloride solution and sterilizes,
Aseptic water washing, cuts fruit with scalpel, brown powder seed is inoculated on culture medium with transfer needle, cultivates 80-100
My god, form protocorm and bud mixture；
C, Proliferation, Differentiation cultivation stage：To obtain more seedlings, bud and protocorm line and staff control are seeded in 1/2MS additions 6-
Benzylaminopurine (6-BA), α-naphthylacetic acid（NAA）On culture medium, cultivated through 140-160 days, grow up to grow thickly it is small with short
Seedling；
D, strengthening seedling and rooting stage：The clustered plants grown up to are separated, 1/4MS addition α-naphthylacetic acids are inoculated into（NAA）Culture medium
On, the intact plant until growing up to tool 2-3 bars root, 3-4 piece leaves.
E, hardening and transplanting：Refined under natural lighting before the bottle outlet when test tube seedling length is to 3-4 centimetres, bottle outlet in greenhouse
Seedling, then takes out it from vial, moves into broken bark and mixed-matrix humous；Strengthen temperature, temperature, shelter from heat or light and
Ventilating management, " excellent pocket blue " can normal growth, the transplanting survival rate of seedling is up to more than 90%；Periodically pour once it is nitrogenous,
The composite fertilizer of phosphorus, potassium, thin fertile duty is applied, and the Lanzhou-Xinjiang kind of pocket " excellent pocket is blue " kind can be set up by strengthening pest and disease damage pipe irrigation project in cultivation
The industrialization breeding method system of seedling.
The beneficial effects of the invention are as follows：" excellent pocket is blue " combines the merit of parents, and compact with plant type, flower-shape is only
Special elegant, pattern is splendid beautiful, it is easy to cultivate, and the average florescence, what it maintained female parent spent more characteristic and male parent up to 50 days
Short scape characteristic, it is to avoid the long easy lodging of several single and scape and the shortcoming fractureed are spent, with high ornamental value.Seed
Germination rate and flowering rate are high, and cultivation period is 3-4, are the treasures of great ornamental value in the Lanzhou-Xinjiang product of pocket, and the present invention passes through pocket
The tissue culture propagation method research of Lanzhou-Xinjiang kind " excellent pocket is blue ", can improve breeding coefficient, be to obtain a large amount of " excellent pocket is blue " to plant
The high efficiency method for test tube propagation of seedling, high with germination rate, seedling is fast, the advantage such as seedling quality better, growth potential are strong, can obtain
Ordered one test tube seedling, and provide substantial amounts of seedling in a short time, provides one and has for the seeling industry of " excellent pocket is blue "
The approach of effect, so as to preferably protect wild resource.
Brief description of the drawings
The brief description of the drawings of the present invention is as follows：
Fig. 1：Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " picture of blooming；
Fig. 2：Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " cultivation picture.
Embodiment
The present invention is further illustrated below：
Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method, comprises the following steps：
A, artificial hybridization pollination：It is female parent with long valve pocket orchid, leaflet pocket orchid is male parent, the long valve pocket for choosing robust growth is blue female
This and male parent carry out artificial supplementary pollination when blooming, 370 to 390 days after pollination, will be developed to and are mature on the whole and uncracked
Fruit is used as explant；
B, aseptic seeding and protocorm sprout the stage：From full fruit with alcohol-pickled, disappear in the mercuric chloride solution being placed in
Poison, aseptic water washing cuts fruit with scalpel, brown powder seed is inoculated on culture medium with transfer needle, cultivates 80-
100 days, form protocorm and bud mixture；
C, Proliferation, Differentiation cultivation stage：To obtain more seedlings, bud and protocorm line and staff control are seeded in 1/2MS additions 6-
Benzylaminopurine (6-BA), α-naphthylacetic acid（NAA）On culture medium, cultivated through 140-160 days, grow up to grow thickly it is small with short
Seedling；
D, strengthening seedling and rooting stage：The clustered plants grown up to are separated, 1/4MS addition α-naphthylacetic acids are inoculated into（NAA）Culture medium
On, the intact plant until growing up to tool 2-3 bars root, 3-4 piece leaves；
E, hardening and transplanting：Natural lighting lower refining seedling before the bottle outlet when test tube seedling length is to 3-4 centimetres, bottle outlet in greenhouse, so
It is taken out from vial afterwards, moved into broken bark and mixed-matrix humous；Strengthen temperature, temperature, shelter from heat or light and divulge information
Management, " excellent pocket blue " can normal growth, the transplanting survival rate of seedling is up to more than 90%；Periodically pour once nitrogenous, phosphorus, potassium
Composite fertilizer, thin fertile duty applied, and the Lanzhou-Xinjiang kind of pocket " excellent pocket is blue " seedling can be set up by strengthening pest and disease damage pipe irrigation project in cultivation
Industrialization breeding method system.
Further, the tissue-cultured seedling of " excellent pocket is blue " is obtained, the crossover process between male parent and female parent is to realize following three
The most important step of individual problem：1st, whether the seed obtained is " excellent pocket is blue " new varieties；2nd, the seed that hybridization is obtained is
It is no to cultivate " excellent pocket is blue "；Whether " excellent pocket is blue " the 3rd, cultivated reaches quality requirements.Specifically way is：August
The first blue season of flowers of pocket, the artificial situation of blooming for controlling to adjust Parent, when maternal long valve pocket orchid bud opens soon, sprinkling is once
Concentration promotes long valve pocket orchid to bloom for 1000PPM ethephon (CEPHA),2-(chloroethyl) phosphonic acid, promotes leaflet pocket orchid to bloom with same method again after one week,
Bloomed with long valve pocket orchid and carry out artificial supplementary pollination again after starting the clock two weeks, the orchid secretion stigma of this duration valve pocket is largely glued
Liquid, leaflet pocket orchid powder is vigourous, and hybridization affinity is strong, best results, completely filled fruit, and survival rate is high after seed tissue culture.Pass through
The method, the fruit-setting rate for obtaining " excellent pocket is blue " seed brings up to more than 95%, and maternal, the above-mentioned selection at the florescence of male parent is to carry
The deciding factor of high fruit-setting rate.（It is only 20% due to lacking fruit-setting rate under effective insect pollinator, natural conditions）, and fruit
Full, planting percent is high after seed tissue culture.
Further, described when using alcohol-pickled in step B, from 75% alcohol, soak time is 5min；Rise
Sterilization refers to 0.1% mercuric chloride solution in mercury solution, soaks 10 min；Described sterilized water is distilled water or suds.
Further, in step B, described culture medium refers to that 1/2MS adds 6- benzylaminopurines (6-BA) 0.5-1.0
Mg/litre, α-naphthylacetic acid（NAA0.1-0.2 mg/litre culture mediums.Culture medium be tissue-cultured seedling can be formed and grow extremely weigh
The factor wanted.And the above-mentioned formula and concentration of 6- benzylaminopurines, α-naphthylacetic acid are even more the pass for determining tissue-cultured seedling success or not
Key.
Further, in step B, aseptic seeding and protocorm sprouting stage carry out next step, abortion when cultivating 90 days
Rate is minimum.
Further, in step C in culture medium prescription, 6- benzylaminopurines (6-BA) concentration be 2-4 mg/litres, α-
Methyl α-naphthyl acetate（NAA）Concentration is 0.1-0.5 mg/litres, and incubation time is to be preferred for 150 days.
Further, in step D, clustered plants are inoculated into 1/4MS addition α-naphthylacetic acids after separating（NAA）0.1-0.5 millis
G/l culture medium on, culture grows up to after 55-65 days has 2-3 bars roots, the intact plant of 3-4 piece leaves.
Further, in step E, when test tube seedling length is to 3-4 centimetres, the natural lighting lower refining seedling before bottle outlet in greenhouse
10 days, then it is taken out from vial, clean the culture medium of root, it is 4 to move into broken bark and fertile soil:1 mixed base
In matter.Greenhouse keeps sheltering from heat or light and divulging information for 15-28 DEG C of temperature, more than 80% humidity, 70-80%, and " excellent pocket is blue " can be just
It is frequently grown, the transplanting survival rate of seedling is up to more than 90%.Pour within usual 15 days or so a nitrogen：Phosphorus：The ratio of potassium is 14：16；
1000 times of 15 composite fertilizer, thin fertile duty is applied, and strengthens pest and disease damage pipe irrigation project in cultivation.
Protection scope of the present invention is not limited only in the technical scheme disclosed in embodiment, all present patent applications respectively
The step of technical scheme, the method that tissue-culturing rapid propagation is carried out to " excellent pocket is blue ", and carried out on the basis of above-mentioned technical proposal
Improvement, equivalent substitution, each fall within protection scope of the present invention.
Claims (8)
- Pocket Lanzhou and Xinjiang 1. kind " excellent pocket is blue " tissue culture and rapid propagation method, comprises the following steps：A, artificial hybridization pollination：It is female parent with long valve pocket orchid, leaflet pocket orchid is male parent, the long valve pocket for choosing robust growth is blue female This and male parent carry out artificial supplementary pollination when blooming, 370 to 390 days after pollination, will be developed to and are mature on the whole and uncracked Fruit is used as explant；B, aseptic seeding and protocorm sprout the stage：From full fruit with alcohol-pickled, disappear in the mercuric chloride solution being placed in Poison, aseptic water washing cuts fruit with scalpel, brown powder seed is inoculated on culture medium with transfer needle, cultivates 80- 100 days, form protocorm and bud mixture；C, Proliferation, Differentiation cultivation stage：To obtain more seedlings, bud and protocorm line and staff control are seeded in 1/2MS additions 6- Benzylaminopurine (6-BA), α-naphthylacetic acid（NAA）On culture medium, cultivated through 140-160 days, grow up to grow thickly it is small with short Seedling；D, strengthening seedling and rooting stage：The clustered plants grown up to are separated, 1/4MS addition α-naphthylacetic acids are inoculated into（NAA）Culture medium On, the intact plant until growing up to tool 2-3 bars root, 3-4 piece leaves；E, hardening and transplanting：Natural lighting lower refining seedling before the bottle outlet when test tube seedling length is to 3-4 centimetres, bottle outlet in greenhouse, so It is taken out from vial afterwards, moved into broken bark and mixed-matrix humous；Strengthen temperature, temperature, shelter from heat or light and divulge information Management, " excellent pocket blue " can normal growth, the transplanting survival rate of seedling is up to more than 90%；Periodically pour once nitrogenous, phosphorus, potassium Composite fertilizer, thin fertile duty applied, and the Lanzhou-Xinjiang kind of pocket " excellent pocket is blue " seedling can be set up by strengthening pest and disease damage pipe irrigation project in cultivation Industrialization breeding method system.
- The Lanzhou-Xinjiang kind of pocket according to claim 1 2. " excellent pocket is blue " tissue culture and rapid propagation method, it is characterised in that：The first pocket of August Blue season of flowers, the artificial situation of blooming for controlling to adjust Parent when maternal long valve pocket orchid bud opens soon, sprays a concentration Promote long valve pocket orchid to bloom for 1000PPM ethephon (CEPHA),2-(chloroethyl) phosphonic acid, promote leaflet pocket orchid to bloom with same method again after one week, with length Valve pocket orchid blooms start the clock two weeks after carry out artificial supplementary pollination again, this duration valve pocket orchid secretion stigma a large amount of mucus, Leaflet pocket orchid powder is vigourous, and hybridization affinity is strong, best results, completely filled fruit, and survival rate is high after seed tissue culture.
- The Lanzhou-Xinjiang kind of pocket according to claim 1 3. " excellent pocket is blue " tissue culture and rapid propagation method, it is characterised in that：In step B, Described when using alcohol-pickled, from 75% alcohol, soak time is 5min；Sterilization refers to 0.1% mercuric chloride in mercuric chloride solution Solution, soaks 10 min；Described sterilized water is distilled water or suds.
- The Lanzhou-Xinjiang kind of pocket according to claim 1 4. " excellent pocket is blue " tissue culture and rapid propagation method, it is characterised in that：In step B, Described culture medium refers to 1/2MS addition 6- benzylaminopurine (6-BA) 0.5-1.0 mg/litres, α-naphthylacetic acid（NAA0.1- 0.2 mg/litre culture medium.
- The Lanzhou-Xinjiang kind of pocket according to claim 1 5. " excellent pocket is blue " tissue culture and rapid propagation method, it is characterised in that：In step B, Aseptic seeding and protocorm sprouting stage carry out next step when cultivating 90 days.
- The Lanzhou-Xinjiang kind of pocket according to claim 1 6. " excellent pocket is blue " tissue culture and rapid propagation method, it is characterised in that：In step C In culture medium prescription, 6- benzylaminopurines (6-BA) concentration is 2-4 mg/litres, α-naphthylacetic acid（NAA）Concentration is 0.1-0.5 Mg/litre, incubation time is to be preferred for 150 days.
- The Lanzhou-Xinjiang kind of pocket according to claim 1 7. " excellent pocket is blue " tissue culture and rapid propagation method, it is characterised in that：In step D, Clustered plants are inoculated into 1/4MS addition α-naphthylacetic acids after separating（NAA）On the culture medium of 0.1-0.5 mg/litres, 55-65 is cultivated Grow up to tool 2-3 bars root, the intact plant of 3-4 piece leaves after it.
- 8. the Lanzhou-Xinjiang kind of pocket " excellent pocket is blue " tissue culture and rapid propagation method according to claim 1-7 any claims, it is special Levy and be：In step E, when test tube seedling length is to 3-4 centimetres, the natural lighting lower refining seedling before bottle outlet in greenhouse 10 days, then It is taken out from vial, the culture medium of root is cleaned, it is 4 to move into broken bark and fertile soil:In 1 mixed-matrix；Greenhouse Keep 15-28 DEG C of temperature, more than 80% humidity, 70-80% to shelter from heat or light and divulge information, " excellent pocket is blue " can normal growth, into The transplanting survival rate of seedling is up to more than 90%；Pour within usual 15 days or so a nitrogen：Phosphorus：The ratio of potassium is 14：16；15 composite fertilizer 1000 times, thin fertile duty is applied, and strengthens pest and disease damage pipe irrigation project in cultivation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710472440.0A CN107079817A (en) | 2017-06-21 | 2017-06-21 | Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method |
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| CN201710472440.0A CN107079817A (en) | 2017-06-21 | 2017-06-21 | Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108770680A (en) * | 2018-05-23 | 2018-11-09 | 广东省农业科学院环境园艺研究所 | A kind of method of the sapling multiplication and cultivation of red rosy clouds pocket orchid |
| CN109258463A (en) * | 2018-09-18 | 2019-01-25 | 广西壮族自治区林业科学研究院 | A kind of asexual reproduction method of paphiopedilum armeniacum |
| CN110089428A (en) * | 2019-04-10 | 2019-08-06 | 福建省农业科学院作物研究所 | A kind of pocket orchid hybrid seed aseptic seeding growing seedlings method |
| CN110149950A (en) * | 2019-06-06 | 2019-08-23 | 广西壮族自治区中国科学院广西植物研究所 | Improve the method and its test solution of the sub- development quality of white flower pocket orchid species |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108770680A (en) * | 2018-05-23 | 2018-11-09 | 广东省农业科学院环境园艺研究所 | A kind of method of the sapling multiplication and cultivation of red rosy clouds pocket orchid |
| CN109258463A (en) * | 2018-09-18 | 2019-01-25 | 广西壮族自治区林业科学研究院 | A kind of asexual reproduction method of paphiopedilum armeniacum |
| CN109258463B (en) * | 2018-09-18 | 2022-08-02 | 广西壮族自治区林业科学研究院 | Vegetative propagation method of paphiopedilum armeniacum |
| CN110089428A (en) * | 2019-04-10 | 2019-08-06 | 福建省农业科学院作物研究所 | A kind of pocket orchid hybrid seed aseptic seeding growing seedlings method |
| CN110149950A (en) * | 2019-06-06 | 2019-08-23 | 广西壮族自治区中国科学院广西植物研究所 | Improve the method and its test solution of the sub- development quality of white flower pocket orchid species |
| CN110149950B (en) * | 2019-06-06 | 2021-12-03 | 广西壮族自治区中国科学院广西植物研究所 | Method for improving development quality of paphiopedilum albolanum seeds and test solution thereof |
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